Trpm-2 antisense therapy

ABSTRACT

It has now been determined that antisense therapy which reduces the expression of TRPM-2 provides therapeutic benefits in the treatment of cancer. Addition of antisense TRPM-2 ODN to prostatic tumor cells in vivo is effective for delaying the onset of androgen independence. Combined use of antisense TRPM-2 and taxanes synergistically enhances cytotoxic chemosensitivity of androgen-independent prostate cancer. In addition, it has also been found that antisense TRPM-2 has beneficial effect for other cancer types. Specifically, antisense TRPM-2 ODN enhances chemosensitivity in human Renal cell cancer, a normally chemoresistant disease with no active chemotherapeutic agent having an objective response rate higher than 10%. Radiation sensitivity is also enhanced when cells expressing TRPM-2 are treated with antisense TRPM-2 ODN. Thus, the antisense TRPM-2 ODNs can be used to enhance hormone sensitivity, chemosensitivity and radiation sensitivity of a variety of cancer types in which expression of TRPM-2 has been observed.

This application is a continuation of U.S. patent application Ser. No.09/913,325 filed Aug. 10, 2001, which is a Section 371 National Phaseapplication of PCT/US00/04875 and claims the benefit of U.S. ProvisionalPatent Application No. 60/121,726, filed Feb. 26, 1999, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

This application relates to antisense treatments for cancer making useof an antisense oligonucleotide that binds to testosterone-repressedprostate message-2 (TRPM-2).

Prostate cancer is the most common cancer that affects men, and thesecond leading cause of cancer deaths in men in the Western world.Because prostate cancer is an androgen-sensitive tumor, androgenwithdrawal, for example via castration, is utilized in some therapeuticregimens for patients with advanced prostate cancer. Androgen withdrawalleads to extensive apoptosis in the prostate tumor, and hence to aregression of the disease. However, castration-induced apoptosis is notcomplete, and a progression of surviving tumor cells toandrogen-independence ultimately occurs. This progression is the mainobstacle to improving survival and quality of life, and efforts havetherefore been made to target androgen-independent cells. These effortshave focused on non-hormonal therapies targeted againstandrogen-independent tumor cells (Yagoda et al., Cancer 71 (Supp. 3):1098-1109 (1993); Oh et al., J. Urol. 60: 1220-1229 (1998)), however, sofar no non-hormonal agent has improved survival. Alternative approachesare therefore indicated.

It has been observed that numerous proteins are expressed in increasedamounts by prostate tumor cells following androgen withdrawal. At leastsome of these proteins are assumed to be associated with the observedapoptotic cell death which is observed upon androgen withdrawal. (Raffoet al., Cancer Res.: 4448-4445 (1995); Krajewska et al., Am. J. Pathol.148: 1567-1576 (1996); McDonnell et al., Cancer Res. 52: 6940-6944(1992)). The functions of many of the proteins, however, is not clearlyunderstood. TRPM-2 (also known as sulfated glycoprotein-2 (SGP-2) orclusterin) is within this latter category.

TRPM-2 is a ubiquitous protein, with a diverse range of proposedactivities. In prostate epithelial cell, expression of TRPM-2 increasesimmediately following castration, reaching peak levels in rat prostatecells at 3 to 4 days post castration, coincident with the onset ofmassive cell death. These results have led some researchers to theconclusion that TRPM-2 is a marker for cell death, and a promoter ofapoptosis. On the other hand, the observation that Sertoli cells andsome epithelial cells express high levels of TRPM-2 without increasedlevels of cell death, raises questions as to whether this conclusion iscorrect.

Sensibar et al., Cancer Research 55: 2431-2437 (1995) reported on invitro experiments performed to more clearly elucidate the role of TRPM-2in prostatic cell death. They utilized LNCaP cells transfected with agene encoding TRPM-2 and observed whether expression of this proteinaltered the effects of tumor necrosis factor α (TNFα), to which LNCaPcells are very sensitive, with cell death normally occurring withinabout 12 hours. Treatment of the transfected LNCaP cells with TNFα wasshown to result in a transient increase in TRPM-2 levels for a period ofa few hours, but these levels had dissipated by the time DNAfragmentation preceeding cell death was observed. Using an antisensemolecule corresponding to the bases 1-21 of the TRPM-2 sequence, but notother TRPM-2 antisense oligonucleotides, resulted in a substantialreduction in expression of TRPM-2, and an increase in apoptotic celldeath in LNCaP cells exposed to TNFα. This led Sensibar et al. to thehypothesis that overexpression of TRPM-2 could protect cells from thecytotoxic effect of TNF, and that TRPM-2 depletion is responsible forthe onset of cell death, although the mechanism of action remainsunclear.

While Sensibar et al. provides information about the possible role ofTRPM-2, it nevertheless discloses results from only a model system inwhich expression of TRPM-2 is based on a transfected gene. Furthermore,expression levels of TRPM-2 is very low or absent in LNCaP cells grownin other labs. The situation which results in vivo when prostate tumorcells are subjected to androgen withdrawal is far more complex, withnumerous proteins changing expression levels as a result. Thus, it isnot possible from the Sensibar et al. data to predict whether TRPM-2would perform the same function when present in combination with otherproteins, or whether changes in levels of TRPM-2 following androgenwithdrawal in vivo could provide any therapeutic benefits. Indeed, thefact that TRPM-2 is expressed in substantial quantities in prostatictumor cells at various stages following androgen withdrawal, includingstages where significant apoptotic cell death is occurring suggests thatrole of TRPM-2 in vivo may be more complicated. Thus, while the artprovides data concerning certain aspects of apoptotic cell death inprostatic tumor cells, it offers neither a teaching or a suggestion of amethodology to provide a delay in the onset of androgen-independence.

It is an object of the present invention to provide such a method.

It is a further object of the present invention to provide therapeuticantisense molecules for delaying the onset of androgen independence inprostatic tumor cells.

It is an additional object of the present invention to provide a methodfor enhancing the chemosensitivity or radiation sensitivity of cancercells from a cancer that expresses TRPM-2.

It is a further object of the present invention to provide therapeuticantisense molecules for inhibiting expression of TRPM-2.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has now been determinedthat antisense therapy which reduces the expression of TRPM-2 providestherapeutic benefits in the treatment of cancer. In particular, suchantisense therapy can be applied in treatment of prostate cancer andrenal cell cancer.

Addition of antisense TRPM-2 oligodeoxynucleotide (ODN) to prostatictumor cells in vivo is effective for delaying the onset of androgenindependence. Thus, in one aspect, the invention provides a method fortreating prostate cancer in an individual suffering from prostatecancer, comprising the steps of initiating androgen-withdrawal to induceapoptotic cell death of prostatic tumor cells in the individual, andadministering to the individual a composition effective to inhibitexpression of TRPM-2 by the tumor cells, thereby delaying theprogression of prostatic tumor cells to an androgen-independent state inan individual. Furthermore, combined use of antisense TRPM-2 pluscytotoxic chemotherapy (e.g. taxanes) synergistically enhanceschemosensitivity in hormone refractory prostate cancer. In anotheraspect of the invention, a second antisense ODN which inhibitsexpression of an anti-apoptotic protein other than TRPM-2 isadministered along with the antisense TRPM-2 ODN.

It has also been found that antisense TRPM-2 has beneficial effects forother cancer types. Specifically, antisense TRPM-2 ODN enhanceschemosensitivity in human Renal cell cancer, a normally chemoresistantdisease with no active chemotherapeutic agent having an objectiveresponse rate higher than 10%. Radiation sensitivity is also enhancedwhen cells expressing TRPM-2 are treated with antisense TRPM-2 ODN.Thus, the antisense TRPM-2 ODNs can be used to treat a variety of cancertypes in which expression of TRPM-2 has been observed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the delay in onset of androgen-independence which isachieved using an antisense TRPM-2 ODN;

FIG. 2 shows the positions of 10 antisense oligonucleotides evaluatedfor the ability to inhibit TRPM-2 expression and delay onset ofandrogen-independence;

FIG. 3 shows expression levels of TRPM-2 mRNA in the presence of variousantisense ODNs;

FIG. 4 shows the levels of TRPM-2 mRNA in Shionogi cells treated invitro with varying amounts of antisense TRPM-2 ODN or a mismatchcontrol;

FIG. 5 shows the dose-response curve for combinations of taxol andantisense TRPM-2 ODN;

FIG. 6 shows the dose-response curve for combinations of taxol,antisense TRPM-2 ODN and antisense Bcl-2 ODN;

FIG. 7A shows decease in TRPM-2 mRNA levels in human renal cell cancerafter treatment with antisense TRPM-2 ODNs;

FIG. 7B shows the increase in chemosensitivity of human renal cellcancer to taxol after treatment with antisense TRPM-2 ODNs;

FIG. 8 shows TRPM-2 expression in PC-3 prostate cancer cells aftervarious doses of radiation;

FIGS. 9A and 9B show the comparative radiation resistance of humanprostate cell lines which overexpress (LNCaP/T) and normally (LNCaP/P)express TRPM-2;

FIG. 10 shows the increased susceptibility of PC-3 cells to radiationafter treatment with antisense TRPM-2 ODN; and

FIGS. 11A and 11B show the increased sensitivity of PC-3 cells toradiation after treatment with antisense TRPM-2 ODN.

FIGS. 12A and 128 show the increased sensitivity of Shionogi tumor cellsto chemotherapy agents paclitaxel and mitoxanthrone when administeredwith antisense TRPM-2 ODN.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antisense TRPM-2 ODNs and to the use ofthese compositions in the treatment of cancer. The invention can beapplied in the treatment of cancers where the cancer cells expressTRPM-2. Three significant classes of cancer cells which express TRPM-2are prostate cancer cells, human renal cell cancer (RCC) cells and somebreast cancer cells.

In one embodiment, the present invention provides a method for enhancingcastration-induced tumor cell death and delaying the progression ofprostatic tumor cells to androgen independence; a therapeutic method forthe treatment of individuals, including humans, suffering from prostatecancer; and therapeutic agents effective for use in such methods. Thetherapeutic method of the invention will most commonly be used in thetreatment of individuals with advanced prostate cancer.

Enhancement of castration-induced tumor cell death and delay of theprogression of androgen-sensitive prostatic cancer cells toandrogen-independent is achieved by inhibiting the expression of TRPM-2by the cells. Experiments were performed in three model systems, the invivo Shionogi tumor model, the human TRPM-2 transfected LNCaP model, andthe human PC-3 model, which taken together demonstrated that suchinhibition leading to delay of androgen-independence can be achieved bytreating androgen-sensitive prostatic tumor cells with antisenseoligodeoxynucleotides (ODNs).

In the first experiment, the ability of a mouse TRPM-2 antisensemolecule, (Seq. ID. No. 1) to delay onset of androgen independence inthe Shionogi tumor model was evaluated. The Shionogi tumor model is axenograft of an androgen-dependent mouse mammary carcinoma that growssubcutaneously in male syngeneic hosts. Shionogi tumor cells are highlytumorigenic and locally invasive. The cells have been shown to respondto androgen withdrawal in a manner which mimics the observed behavior ofprostatic tumor cells, and have been accepted as a valid model forprostate cancer in humans. (Bruchovsky et al., Cancer Res. 50: 2275-2282(1990); Rennie et al., Cancer Res. 48: 6309-6312 (1988); Bruchovsky etal., Cell 13: 272-280 (1978); Gleave et al., in Genitourinary Oncology,pp. 367-378, Lange et al., eds, Lippencott (1997); Cleave et al., J.Urol. 157: 1727-1730 (1997); Bruchovsky et al., The Prostate 6: 13-21(1996)). Thus, androgen withdrawal precipitates apoptosis and tumorregression in a highly reproducible mariner. Further, changes inexpression of TRPM-2 and Bcl-2 in human prostate cancer followingcastration and during progression to androgen independence are similarto those observed in Shionogi tumor cells. Thus, the Shionogi tumormodel mimics many of the characteristics of prostate cancer cells.Further, the Shionogi tumor model provides a very useful model for theevaluation of the ability of compounds to delay the onset ofandrogen-independence. Despite complete tumor regression aftercastration, rapidly growing androgen-independent Shionogi tumorsinvariably recur after one month, which provides a reliable end point toevaluate agents which can delay the progression toandrogen-independence. In general, events which occur in the Shionogitumor model within one month occur in human patients within about twoyears.

The ability of the antisense ODNs that inhibit expression of TRPM-2 todelay the onset of androgen-independence was evaluated by measuringtumor volume post-castration in the Shionogi tumor model. The testanimals (n=7) were treated intraperitoneally once daily with 12.5 mg/kgrepeat doses of antisense TRPM-2 ODNs (Seq. ID. No 1) in a bufferedsaline solution. As a control, animals (n=7) were treated with amismatch ODN (Seq. ID. No. 2). As shown in FIG. 1, both test and controlgroups showed the expected decline in tumor volume immediately followingcastration, but the tumors in the antisense TRPM-2 ODN-treated miceregressed faster than the controls. The control group also exhibited theexpected increase in tumor volume which is associated the development ofandrogen-independence. In contrast, at 49 days post-castration, littletumor regrowth had occurred in the mice treated using the antisenseTRPM-2 ODN. Tumors did eventually recur in the antisense TRPM-2ODN-treated mice, but the median time to recurrence is approximatelytwice that of the control group. Thus, inhibition of TRPM-2 is effectivenot only for increasing the amount of cell death which occursimmediately following androgen withdrawal, but also for delaying theonset of androgen-independence. The more rapid decrease in tumor volumein the mice treated with antisense TRPM-2 ODNs was due to earlier onsetand more extensive castration-induced apoptosis. This was confirmed bydetecting poly(ADP-ribose) polymerase (PARP) cleavage fragments inShionogi tumor specimens (Miyake, et al., Cancer Res. 60:170-176(2000)).

To evaluate which human antisense ODNs complementary to TRPM-2 mRNAsequences are most effective for this purpose, a series of ten antisensephosphorothioate ODNs were prepared spanning various mRNA regions asshown in FIG. 2. The sequences of these ten ODNs are set forth in theattached Sequence Listing as Seq. ID. Nos. 3-12. The ten human antisenseODNs were evaluated using TRPM-2 transfected LNCaP cells and humanprostate cancer PC-3 cells for their ability to inhibit expression ofTRPM-2 mRNA As shown in FIG. 3, the antisense ODNs tested producedvariable levels of inhibition of TRPM-2 mRNA expression, with the bestresults being achieved with Seq. ID Nos. 4, 5, and 12. Sequence ID No. 5corresponds to the sequence used by Sensibar et al. that producedinhibition of TRPM-2 expression in LNCaP cells, and is complementary tothe first 21 bases of the TRPM-2 mRNA. The most effectivedown-regulation occurred with Seq. ID No. 4. Common to all of theeffective sequences is an overlap with either the initiation ortermination sites of the TRPM-2 mRNA. Thus, in a general sense, themethod of the invention can be practiced with antisense oligonucleotideswhich are complementary to a region of the TRPM-2 mRNA spanning eitherthe translation initiation site or the termination site.

In accordance with a further aspect of with the invention, therapeutictreatment of individuals, including human individuals, suffering fromprostate cancer is achieved by initiating androgen-withdrawal to induceapoptotic cell death of prostatic tumor cells in the individual, andadministering to the individual a composition effective to inhibitexpression of TRPM-2 by the tumor cells, thereby delaying theprogression of prostatic tumor cells to an androgen-independent state inan individual.

Initiation of androgen withdrawal may be accomplished via surgical(removal of both testicles) or medical (drug-induced suppression oftestosterone) castration, which is currently indicated for treatment ofprostate cancer. Medical castration can be achieved by various regimens,including LHRH agents or antiandrogens. (Gleave et al., CMAJ 160:225-232 (1999)). Intermittent therapy in which reversible androgenwithdrawal is effected is described in Gleave et al. Eur. Urol. 34(Supp. 3): 37-41 (1998).

The inhibition of TRPM-2 expression may be transient, and ideally shouldoccur coincident with androgen withdrawal. In humans, this means thatinhibition of expression should be effective starting within a day ortwo of androgen withdrawal and extending for about 3 to 6 months. Thismay require multiple doses to accomplish. It will be appreciated,however, that the period of time may be more prolonged, starting beforecastration and expending for substantial time afterwards withoutdeparting from the scope of the invention.

Antisense TRPM-2 ODNs have also been determined to enhancechemosensitivity in human renal cell cancer (RCC). RCC is achemoresistant disease with no active chemotherapeutic agent withobjective response rates higher than 10%. Increased TRPM-2 expression inrenal proximal convoluted cells undergoing apoptosis has been observedafter various stimuli including ureteral obstruction andaminoglycosides. However, functional significance of TRPM-2 expressionin RCC has not been well documented. Test results show, however, thatantisense TRPM-2 ODN enhances chemosensitivity in human RCC CaKi-2 cells(See Example 6, infra).

Antisense TRPM-2 ODNs were also found to increase sensitivity toradiation (See Example 7 and FIG. 8).

Inhibition of expression of TRPM-2 may be accomplished by theadministration of antisense ODNs, particularly antisense ODNs which arecomplementary to a region of the TRPM-2 mRNA spanning either thetranslation initiation site or the termination site. For treatment ofprostate cancer in humans, specific useful sequences are those shown inSeq. ID Nos. 4, 5 and 12.

The ODNs employed may be modified to increase the stability of the ODNin vivo. For example, the ODNs may be employed as phosphorothioatederivatives (replacement of a non-bridging phosphoryl oxygen atoms witha sulfur atom) which have increased resistance to nuclease digestion.MOE (2′-O-(2-methoxyethyl) modification (ISIS backbone) is alsoeffective.

Administration of antisense ODNs can be carried out using the variousmechanisms known in the art, including naked administration andadministration in pharmaceutically acceptable lipid carriers. Forexample, lipid carriers for antisense delivery are disclosed in U.S.Pat. Nos. 5,855,911 and 5,417,978 which are incorporated herein byreference. In general, the antisense is administered by intravenous,intraperitoneal, subcutaneous or oral routes, or direct local tumorinjection. From the experiments performed using the Shionogi mousemodel, it appears that the antisense ODN is preferentially active in thetumor cells. Indeed, TRPM-2 expression in non-tumor tissues wassubstantially unaffected, and no side effects of the antisense ODNadministration were observed.

The amount of antisense ODN administered is one effective to inhibit theexpression of TRPM-2 in prostatic cells. It will be appreciated thatthis amount will vary both with the effectiveness of the antisense ODNemployed, and with the nature of any carrier used. The determination ofappropriate amounts for any given composition is within the skill in theart, through standard series of tests designed to assess appropriatetherapeutic levels.

The method for treating prostate cancer in accordance with the inventionmay further include administration of chemotherapy agents and/oradditional antisense ODNs directed at different targets. For example, ithas been found using the Shionogi tumor model that antisense TRPM-2 ODNincreases sensitivity to conventional chemotherapy agents such astaxanes (paclitaxel or docetaxel) and mitoxanthrone (FIGS. 12A and 12B).As shown in FIGS. 12A and 12B, treatment with antisense TRPM-2 ODN inthe presence of taxol or mitoxanthrone resulted in a reduced tumorvolume as compared to the combination of taxol or mitoxanthrone with themismatch (MM) ODN. Other agents likely to show synergistic activityinclude other cytotoxic agents (e.g. cyclophosphamide, topoisomeraseinhibitors), angiogenesis inhibitors, differentiation agents and signaltransduction inhibitors. Similarly, combinations of TRPM-2 antisensewith other antisense species such as antisense Bcl-2 ODN worked betterat killing Shionogi cells in vitro than either ODN alone. Thus, TRPM-2can work in concert with other antisense molecules, such as antisenseBcl-2, Bcl-x1 and c-myc ODN to provide greater effectiveness.

The invention will now be further described with reference to thefollowing, non-limiting examples.

Example 1

Shionogi tumor model experiments were performed using cells from theToronto subline of transplantable SC-115 AD mouse mammary carcinoma. Forin vivo studies, approximately 5×10⁶ cells of the Shionogi carcinomawere injected subcutaneously in adult male DD/S strain mice. When theShionogi tumors became 1 to 2 cm in diameter, usually 2 to 3 week afterinjection, castration was performed through an abdominal incision undermethoxyflurane anesthesia. Details of the maintenance of mice, tumorstock and operative procedures have been previously described,Bruchovsky et al., Cancer res. 50: 2275-2282 (1990); Rennie et al.,Cancer Res. 48: 6309-6312 (1988); Bruchovsky et al., Cell 13: 272-280(1978); Gleave et al., in Genitourinary Oncology, pp. 367-378, Lange etal., eds, Lippencott (1997); Gleave et al., J. Urol. 157: 1727-1730(1997); Bruchovsky et al., The Prostate 6: 13-21 (1996)).

Mice were randomly selected for treatment with murine phosphorothioateantisense TRPM-2 ODN (Seq. ID No. 1) or a mismatch control (Seq. ID No.2) which is two bases different in sequence from the antisense TRPM-2ODN. Each experimental group consisted of 7 mice. One day aftercastration, 12.5 mg/kg of antisense TRPM-2 or mismatch control ODNdissolved in phosphate buffered saline were injected intraperitoneallyonce daily into each mouse of 40 days. Tumor volume was measured twiceweekly, and calculated by the formula length X width X depth X 0.5236.Gleave et al., Cancer Res. 52: 1598-1605 (1992). Data points werereported as average tumor volumes ±standard deviation.

The results of this study are shown in FIG. 1. As shown, Shionogi tumorsregressed faster and complete regression occurred earlier in micetreated with antisense TRPM-2 ODN. Furthermore, treatment with antisenseTRPM-2 ODN substantially delayed the onset of androgen-independencewhich is reflected by the increase in tumor volume after day 21 in thecontrol animals. No side effects associated with antisense TRPM-2 or themismatch control were observed.

To examine the effects of in vivo ODN treatment on levels of TRPM-2mRNA, Northern blot analysis was performed on Shionogi tumor tissue frommice. The mice were treated daily with 12.5 mg/kg of antisense TRPM-2ODN (n5) or the mismatch control (n=6) by intraperitoneal injectionstarting one day after castration. On the fourth day after castration,tumor tissues were harvested and analyzed by Northern blot for TRPM-2mRNA. Antisense TRPM-2 ODN resulted in a 75% reduction in TRPM-2 mRNAlevels in Shionogi tumors compared to mismatch control ODN treatedtumors. (FIG. 3).

Comparable analyses were performed on normal mouse organs. Samples ofspleen, kidney, prostate and brain were harvested from Shionogi tumormice treated with antisense TRPM-2 ODN and mismatch control under thesame treatment schedule, and analyzed by Northern blot. Although TRPM-2mRNA levels was significantly lower in tumor tissues, antisense TRPM-2ODN had no effect on TRPM-2 mRNA levels in the normal organs.

Example 2

The sequence selectivity of the antisense TRPM-2 ODN (Seq. ID. No. 1)was confirmed by comparing expression levels of TRPM-2 mRNA in Shionogitumor cells maintained in vitro, after treatment with the varying levelsof antisense TRPM-2 ODN or a mismatch control (Seq. ID. No. 2). Tofacilitate uptake of the ODNs into the cells, the ODNs were formulatedin a cationic lipid carrier (Lipofectin™, (Life Technologies, Inc.)).Cells were treated twice over a period of two days using the followingprotocol. Cells were preincubated for 20 minutes with 4 μg/ml oflipofectin in serum free OPTI-MEM™ (Life Technologies, Inc.) and thenincubated with the medium containing the selected concentration of ODNand lipofectin for four hours. The medium was then replaced with thestandard culture medium.

The amount of TRPM-2 mRNA in the cells was evaluated using Northern blotanalysis. As shown in FIG. 4, treatment of Shionogi cells with antisenseTRPM-2 ODN reduced TRPM-2 mRNA levels in a dose dependent manner. Incontrast, TRPM-2 mRNA levels were not affected by the mismatch ODN (Seq.ID. No. 2) at any of the employed concentrations. Thus, the affect ofantisense TRPM-2 ODN is apparently sequence specific.

Example 3

Shionogi cells maintained in vitro were treated with varying amounts oftaxol alone or in combination with 500 nM antisense TRPM-2 ODN (Seq. ED.No. 1) or the mismatch control (Seq. ID No. 2). The cells were treatedtwice, as described in Example 2, and the percentage of viable cellsremaining was determined. The results are summarized in FIG. 5. Asshown, the inclusion of antisense TRPM-2 ODN shined the dose-responsecurve to the left, lowering the IC₅₀ by a factor of 5 to 10. Similarresults were achieved using mitoxanthrone in place of paclitaxel (FIGS.12A and 12B).

Example 4

The experiment of Example 3 was repeated, with the addition of antisenseBcl-2 ODN (Seq. ID. No. 13) or a mismatch Bcl-2 ODN (Seq. ID. No. 14) invarious combinations with antisense/mismatch TRPM-2 ODN and taxol. Theresults are shown in FIG. 6. The combination of antisense TRPM-2 ODNwith antisense Bcl-2 ODN and taxol further enhanced the cytotoxiceffects of taxol. Thus, the targeting of additional anti-apoptoticagents appears to provide therapeutic benefits.

Example 5

To identify appropriate antisense TRPM-2 ODN sequences for use in humantherapy, antisense ODN sequences directed against 10 different sites ofthe human TRPM-2 gene (FIG. 2, Seq. ID Nos. 3-12) were synthesized andtested for their ability to decrease TRPM-2 gene expression in humanprostate cancer PC-3 and transfected LNCaP cells that overexpress TRPM-2using the same treatment protocol described in Example 2. The resultsare summarized in FIG. 3. As shown, sequences 4, 5 and 12 are active forreduction of TRPM-2 expression. These three sequences overlap or areimmediately adjacent to the translation initiation or termination sites.

Example 6

Immunohistochemical staining was used to characterize clusterinexpression in 17 RCC and normal kidney tissues obtained from radicalnephrectomy specimens. TRPM-2 expression in human renal cancer celllines ACHN, CaKi-1 and CaKi-2 was evaluated by Northern and Western blotanalyses. Northern blot analysis was used to assess changes in TRPM-2mRNA expression after antisense TRPM-2 ODN treatment. The effects ofcombined antisense TRPM-2 ODN and taxol treatment on CaKi-2 cell growthwas examined using a MTT assay.

Immunostaining showed an increased clusterin expression in 11 RCCspecimens in comparison to the adjacent normal kidney tissue. In theremaining 6 cases, no difference was seen between malignant and normaltissue. Both TRPM-2 mRNA and protein expression were detectable in allthree human RCC cell lines, with highest levels for CaKi-2.

Antisense TRPM-2 ODN (Seq. ID. No. 1), but not mismatch control ODN(Seq. ID. No. 2), inhibited TRPM-2 expression in CaKi-2 cells in a dosedependant and sequence specific manner (FIG. 7A). Furthermore, antisenseTRPM-2 ODN substantially enhanced taxol chemosensitivity, reducing IC50of taxol by 1 log (500 nM to 50 nM) compared to mismatch control ODN(FIG. 7B). These data demonstrate that TRPM-2 and its protein,clusterin, are expressed at higher levels in RCC compared to normalkidney tissue, and that antisense TRPM-2 ODN may be useful in enhancingthe cytotoxic effects of conventional chemotherapy in advanced RCC.

Example 7

Antisense TRPM-2 ODNs enhance radiation sensitivity of cancer cellswhich express TRPM-2. Using northern analysis, we found that radiationtherapy results in dose and time dependent increases in TRPM2 geneexpression in human prostate cancer PC-3 cells (FIG. 8). Overexpressionof TRPM2 results in increased resistance to radiation induced celldeath. Human prostate LNCaP cells that overexpress TRPM2 (LNCaP/T1) aremore resistant to radiation therapy (FIGS. 9A and B). Treatment of humanprostate cancer PC-3 cells with 100 and 500 nM antisense TRPM-2 ODNs(Seq. ID. NO. 1) significantly reduces cell survival after a singletreatment of 4 Gy radiation therapy compared to mismatch ODN (Seq. IDNo. 2) treatment. (FIG. 10). FIGS. 11A and B show dose dependentradiation sensitization of human prostate cancer PC-3 cells aftertreatment with 10, 50, and 100 nM antisense TRPM-2 oligo in vitro.

Example 8

To determine whether treatment with human antisense TRPM-2 ODN enhanceschemosensitivity in the PC3 human prostate cancer cell line, micebearing PC3 tumors were treated with antisense human TRPM-2 ODN plusmicellar paclitaxel or mitoxantrone, and mismatch control ODN plusmicellar paclitaxel or mitoxantrone (FIGS. 12A and 12B). ODN wasadministered for 28 days and either 0.5 mg micellar taxol or 0.3 mgmitoxantrone were administered on two occasions: from day 10 to 14, andday 24 to 28. A significant reduction in tumor size was observed in theantisense ODN treated animals as compared to those treated with mismatchcontrol ODN. This effect was even more pronounced after the seconddosing of the micellar paclitaxel or mitoxantrone.

1-27. (canceled)
 28. A method for treating an individual suffering froma cancer comprising administering to the individual i) achemotherapeutic agent, and ii) a composition comprising an antisenseoligodeoxynucleotide having nucleotides in the sequence set forth inSeq. ID No. 4 and which oligodeoxynucleotide is modified to increasestability in vivo, wherein the cancer expresses testosterone-repressedprostate message-2 (TRPM-2) in an amount different from normal tissue ofthe same type, thereby treating said individual.
 29. The method of claim28, wherein the chemotherapeutic agent is a taxane.
 30. The method ofclaim 28, wherein the cancer is renal cell carcinoma.
 31. The method ofclaim 28, wherein the cancer is breast cancer.
 32. The method of claim28, wherein the cancer is prostate cancer.
 33. The method of claim 32,wherein the prostate cancer is hormone refractory prostate cancer. 34.The method of claim 28, wherein the taxane is paclitaxel.
 35. The methodof claim 28, wherein the taxane is docetaxel.
 36. The method of claim28, wherein the antisense oligodeoxynucleotide is administered to theindividual intravenously.
 37. The method of claim 28, wherein theantisense oligodeoxynucleotide is administered in a carrier other than alipid carrier.
 38. A method for treating an individual suffering fromprostate cancer and undergoing administration of a taxane comprisingadministering to said individual a composition comprising an antisenseoligodeoxynucleotide having nucleotides in the sequence set forth inSeq. ID No. 4 and which oligodeoxynucleotide is modified to increasestability in vivo, thereby treating said individual.
 39. The method ofclaim 38, wherein the individual is suffering from hormone refractoryprostate cancer.
 40. The method of claim 38, wherein the taxane ispaclitaxel.
 41. The method of claim 38, wherein the antisenseoligodeoxynucleotide is administered to the individual intravenously.42. The method of claim 38, wherein the antisense oligodeoxynucleotideis administered in a carrier other than a lipid carrier.
 43. A methodfor enhancing chemosensitivity in an individual suffering from prostatecancer and undergoing administration of a taxane comprisingadministering to said individual a composition comprising an antisenseoligodeoxynucleotide having nucleotides in the sequence set forth inSeq. ID No. 4 and which oligodeoxynucleotide is modified to increasestability in vivo, thereby enhancing chemosensitivity in saidindividual.
 44. The method of claim 43, wherein the individual issuffering from hormone refractory prostate cancer.
 45. The method ofclaim 43, wherein the taxane is paclitaxel.
 46. The method of claim 43,wherein the antisense oligodeoxynucleotide is administered to theindividual intravenously.
 47. The method of claim 43, wherein theantisense oligodeoxynucleotide is administered other than a lipidcarrier.